Soil conditioner compositions containing lignocellulosic biomass fermentation process syrup

ABSTRACT

Syrup produced in a lignocellulosic biomass fermentation process is used as a binder for soil conditioning materials to make an agricultural composition that is easily handled and applied. The syrup binds powdery soil conditioning materials such as lime and gypsum to form pellets or granules.

This application claims the benefit of United States ProvisionalApplication 62/000639, filed May 20, 2014 and application 61/889061filed Oct. 10, 2013, each of which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates to the field of soil conditioners. Morespecifically, a lignocellulosic syrup is used as a binder incompositions with agricultural application. The lignocellulosic syrup isa co-product of a lignocellulosic biomass fermentation process

BACKGROUND OF THE INVENTION

Soil conditioners are used to improve the quality of soil for improvedplant growth and yield. It is desired that a soil conditioner be appliedin a state that is readily dispersed to the soil. Thus activeingredients of soil conditioners are typically in a finely ground orpowdery form. The conditioning ingredients are typically combined with abinder to form pellets for ease of handling, transportation, anddispersion to application sites.

Examples of soil conditioners include lime which contains calciumcarbonate and other minerals including magnesium, and gypsum which ishydrated calcium sulfate. Lime reduces soil acidity which improves plantgrowth. Gypsum improves soil drainage and promotes plant growth.

An example of a binder that is combined with soil conditioners islignosulfonate (or lignosulfate; sulfonated lignin). Lignosulfonate is aby-product of the sulfite method for manufacturing paper from wood pulp.During this process, lignin in wood is separated from cellulose and issulfonated. The resulting sulfonated lignin by-product is an effectivebinder for powdery substances. WO201426048 discloses a method forproducing a fertilizer wherein pellets of gypsum and a binder, such aslignosulfonate, are made. US20140030369 discloses soil amendmentcompositions containing lignosulfonate.

In a cellulosic ethanol process which makes use of lignocellulosicbiomass as a carbon source for fermentation, whole stillage from adistillation column (beer column) is typically separated into solids(wetcake or filter cake) and liquid (thin stillage) fractions. The thinstillage is passed through evaporators producing a syrup. The filtercake and syrup are co-products of the cellulosic ethanol process. Asyrup with at least about 40% solids may be burned as disclosed inUS20120102823, thereby providing energy. The filter cake may also beburned to provide energy.

There remains a need for additional materials that are readily availablefrom renewable resources, which can be used as binders in soilconditioners.

SUMMARY OF THE INVENTION

The invention provides an agricultural composition that is a combinationof syrup produced from a fermentation process that initially utilizeslignocellulosic biomass and at least one soil conditioning material. Thesyurp of the invention is similar in physical properties to thelignosulfonate materials described above but does not requiresulfonation and is produced from a process that is environmentallyfriendly.

Accordingly, the invention provides an agricultural compositioncomprising:

a) a lignocellulosic syrup; and

b) at least one soil conditioning material.

In one embodiment the syrup is a co-product of a process for theproduction of alcohol from a lignocellulosic biomass.

In another embodiment the syrup comprises:

a) from about 40% to about 70% solids;

b) from about 10 g/l to about 30 g/l of acetamide; and

c) at least about 40 g/l of sugars;

wherein the cellulosic syrup has a density of about 1 to about 2 g/cm3and a viscosity of less than 500 SSU at 100° F. (38° C.). In anotheraspect, the invention provides a process for the production of anagricultural composition comprising combining the lignocellulosic syrupwith at least one soil conditioning material, wherein pellets orgranules comprising the syrup and the conditioning material areproduced.

In yet another aspect, the invention provides a method for conditioningsoil comprising applying an agricultural composition which is describedabove to the soil.

DETAILED DESCRIPTION

To be highly effective, a soil conditioning material is typicallyprepared as a powdery substance that can disperse and penetrate in thesoil to which it is applied. However, for ease of handling andapplication, the powdery substance is combined with a binder to formpellets or granules. A syrup produced from a fermentation process thatuses lignocellulosic biomass may be used as a binder for powdery soilconditioning materials.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, a mixture, process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but may include other elements not expresslylisted or inherent to such composition, mixture, process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

The indefinite articles “a” and “an” preceding an element or componentof the invention are intended to be nonrestrictive regarding the numberof instances (i.e. occurrences) of the element or component. Therefore“a” or “an” should be read to include one or at least one, and thesingular word form of the element or component also includes the pluralunless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures;

through differences in the manufacture, source, or purity of theingredients employed to make the compositions or carry out the methods;and the like. The term “about” also encompasses amounts that differ dueto different equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities. In one embodiment, theterm “about” means within 10% of the reported numerical value,preferably within 5% of the reported numerical value.

The term “fermentable sugar” refers to oligosaccharides andmonosaccharides that can be used as a carbon source by a microorganismin a fermentation process.

The term “lignocellulosic” refers to a composition comprising bothlignin and cellulose. Lignocellulosic material may also comprisehemicellulose.

The term “cellulosic” refers to a composition comprising cellulose andadditional components, including hemicellulose.

The term “saccharification” refers to the production of fermentablesugars from polysaccharides.

The term “pretreated biomass” means biomass that has been subjected topretreatment prior to saccharification. The pretreatment may take theform of physical, thermal or chemical means and combinations thereof.

The term “butanol” refers to isobutanol, 1-butanol, 2-butanol, orcombinations thereof.

The term “lignocellulosic biomass” refers to any lignocellulosicmaterial and includes materials comprising cellulose, hemicellulose,lignin, starch, oligosaccharides and/or monosaccharides. Biomass mayalso comprise additional components, such as protein and/or lipid.Biomass may be derived from a single source, or biomass can comprise amixture derived from more than one source; for example, biomass couldcomprise a mixture of corn cobs and corn stover, or a mixture of grassand leaves. Lignocellulosic biomass includes, but is not limited to,bioenergy crops, agricultural residues, municipal solid waste,industrial solid waste, sludge from paper manufacture, yard waste, woodand forestry waste. Examples of biomass include, but are not limited to,corn cobs, crop residues such as corn husks, corn stover, grasses(including Miscanthus), wheat straw, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sorghum plant material,soybean plant material, components obtained from milling of grains orfrom using grains in production processes (such as DDGS: drieddistillers grains with solubles), woody material such as trees,branches, roots, wood chips, sawdust, shrubs and bushes, leaves,vegetables, fruits, flowers, empty palm fruit bunch, and energy cane.

The term “energy cane” refers to sugar cane that is bred for use inenergy production. It is selected for a higher percentage of fiber thansugar.

The term “lignocellulosic biomass hydrolysate” refers to the productresulting from saccharification of lignocellulosic biomass. The biomassmay also be pretreated or pre-processed prior to saccharification.

The term “lignocellulosic biomass hydrolysate fermentation broth” isbroth containing product resulting from biocatalyst growth andproduction in a medium comprising lignocellulosic biomass hydrolysate.This broth includes components of lignocellulosic biomass hydrolysatethat are not consumed by the biocatalyst, as well as the biocatalystitself and product made by the biocatalyst.

The term “slurry” refers to a mixture of insoluble material and aliquid. A slurry may also contain a high level of dissolved solids.Examples of slurries include a saccharification broth, a fermentationbroth, and a stillage.

The term “whole stillage” refers to the bottoms of a distillation. Thewhole stillage contains the high boilers and any solids of adistillation feed stream. Whole stillage is a type of depleted broth.

The term “thin stillage” refers to a liquid fraction resulting fromsolid/liquid separation of a whole stillage, fermentation broth, orproduct depleted fermentation broth.

The term “syrup” means a concentrated product produced from the removalof water, generally by evaporation, from thin stillage.

The term “target product” refers to any product that is produced by amicrobial production host cell in a fermentation. Target products may bethe result of genetically engineered enzymatic pathways in host cells ormay be produced by endogenous pathways. Typical target products includebut are not limited to acids, alcohols, alkanes, alkenes, aromatics,aldehydes, ketones, biopolymers, proteins, peptides, amino acids,vitamins, antibiotics, and pharmaceuticals.

The term “fermentation” refers broadly to the use of a biocatalyst toproduce a target product. Typically the biocatalyst grows in afermentation broth utilizing a carbon source in the broth, and throughits metabolism produces a target product.

“Solids” refers to soluble solids and insoluble solids. Solids from alignocellulosic fermentation process contain residue from thelignocellulosic biomass used to make hydrolysate medium.

“Volatiles” refers herein to components that will largely be vaporizedin a process where heat is introduced. Volatile content is measuredherein by establishing the loss in weight resulting from heating underrigidly controlled conditions to 950° C. (as in ASTM D-3175). Typicalvolatiles include, but are not limited to, hydrogen, oxygen, nitrogen,acetic acid, and some carbon and sulfur.

“Fixed carbon” refers herein to a calculated percentage made by summingthe percent of moisture, percent of ash, and percent of volatile matter,and then subtracting that percent from 100.

“Ash” is the weight of the residue remaining after burning undercontrolled conditions according to ASTM D-3174.

“Sugars” as referred to in the lignocellulosic syrup composition means atotal of monosaccharide and soluble oligosaccharides.

“Soil conditioning material” refers herein to a substance that improvesthe physical qualities of soil, which may include the ability to providenutrition for plants.

The present agricultural composition contains a lignocellulosic syrupand at least one soil conditioning material.

Production And Composition Of Lignocellulosic Syrup

The present lignocellulosic syrup is produced as a co-product from aprocess that uses lignocellulosic biomass as a source of fermentablesugars which are used as a carbon source for a biocatalyst. Thebiocatalyst uses the sugars in a fermentation process to produce atarget product.

To produce fermentable sugars from lignocellulosic biomass, the biomassis treated to release sugars such as glucose, xylose, and arabinose fromthe polysaccharides of the biomass. Lignocellulosic biomass may betreated by any method known by one skilled in the art to producefermentable sugars in a hydrolysate. Typically the biomass is pretreatedusing physical, thermal and/or chemical treatments, and saccharifiedenzymatically. Thermo-chemical pretreatment methods include steamexplosion or methods of swelling the biomass to release sugars (see forexample WO2010113129; WO2010113130). Chemical saccharification may alsobe used. Physical treatments for pre-processing the biomass include, butare not limited to, grinding, milling, and cutting. Physical treatmentssuch as these may be used for particle size reduction prior to furtherchemical treatment. Chemical treatments include base treatment such aswith strong base (ammonia or NaOH), or acid treatment (US8545633;WO2012103220). In one embodiment the biomass is treated with ammonia(U.S. Pat. No. 7,932,063; U.S. Pat. No. 7,781,191; U.S. Pat. No.7,998,713; U.S. Pat. No. 7,915,017). These treatments release polymericsugars from the biomass. Particularly useful is a low ammoniapretreatment where biomass is contacted with an aqueous solutioncomprising ammonia to form a biomass-aqueous ammonia mixture where theammonia concentration is sufficient to maintain alkaline pH of thebiomass-aqueous ammonia mixture but is less than about 12 weight percentrelative to dry weight of biomass, and where dry weight of biomass is atleast about 15 weight percent solids relative to the weight of thebiomass-aqueous ammonia mixture, as disclosed in U.S. Pat. No.7,932,063, which is herein incorporated by reference.

Saccharification, which converts polymeric sugars to monomeric sugars,may be either by enzymatic or chemical treatments. In one aspect, thepretreated biomass is contacted with a saccharification enzymeconsortium under suitable conditions to produce fermentable sugars.Prior to saccharification, the pretreated biomass may be brought to thedesired moisture content and treated to alter the pH, composition ortemperature such that the enzymes of the saccharification enzymeconsortium will be active. The pH may be altered through the addition ofacids in solid or liquid form. Alternatively, carbon dioxide (CO₂),which may be recovered from fermentation, may be utilized to lower thepH. For example, CO₂ may be collected from a fermenter and fed into thepretreatment product headspace in the flash tank or bubbled through thepretreated biomass if adequate liquid is present while monitoring thepH, until the desired pH is achieved. The temperature is brought to atemperature that is compatible with saccharification enzyme activity, asnoted below. Typically suitable conditions may include temperaturebetween about 40° C. and 50° C. and pH between about 4.8 and 5.8.

Enzymatic saccharification of cellulosic or lignocellulosic biomasstypically makes use of an enzyme composition or blend to break downcellulose and/or hemicellulose and to produce a hydrolysate containingsugars such as, for example, glucose, xylose, and arabinose.Saccharification enzymes are reviewed in Lynd, L. R., et al. (Microbiol.Mol. Biol. Rev., 66:506-577, 2002). At least one enzyme is used, andtypically a saccharification enzyme blend is used that includes one ormore glycosidases. Glycosidases hydrolyze the ether linkages of di-,oligo-, and polysaccharides and are found in the enzyme classificationEC 3.2.1.x (Enzyme Nomenclature 1992, Academic Press, San Diego, Calif.with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995,Supplement 4 (1997) and Supplement 5 [in Eur. J. Biochem., 223:1-5,1994; Eur. J. Biochem., 232:1-6, 1995; Eur. J. Biochem., 237:1-5, 1996;Eur. J. Biochem., 250:1-6, 1997; and Eur. J. Biochem., 264:610-650 1999,respectively]) of the general group “hydrolases” (EC 3.). Glycosidasesuseful in saccharification can be categorized by the biomass componentsthey hydrolyze. Glycosidases useful in saccharification may includecellulose-hydrolyzing glycosidases (for example, cellulases,endoglucanases, exoglucanases, cellobiohydrolases, β-glucosidases),hemicellulose-hydrolyzing glycosidases (for example, xylanases,endoxylanases, exoxylanases, β-xylosidases, arabino-xylanases, mannases,galactases, pectinases, glucuronidases), and starch-hydrolyzingglycosidases (for example, amylases, α-amylases, β-amylases,glucoamylases, α-glucosidases, isoamylases). In addition, it may beuseful to add other activities to the saccharification enzyme consortiumsuch as peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and 3.1.4.x),ligninases (EC 1.11.1.x), or feruloyl esterases (EC 3.1.1.73) to promotethe release of polysaccharides from other components of the biomass. Itis known in the art that microorganisms that producepolysaccharide-hydrolyzing enzymes often exhibit an activity, such as acapacity to degrade cellulose, which is catalyzed by several enzymes ora group of enzymes having different substrate specificities. Thus, a“cellulase” from a microorganism may comprise a group of enzymes, one ormore or all of which may contribute to the cellulose-degrading activity.Commercial or non-commercial enzyme preparations, such as cellulase, maycomprise numerous enzymes depending on the purification scheme utilizedto obtain the enzyme. Many glycosyl hydrolase enzymes and compositionsthereof that are useful for saccharification are disclosed in WO2011/038019. Additional enzymes for saccharification include, forexample, glycosyl hydrolases that hydrolyze the glycosidic bond betweentwo or more carbohydrates, or between a carbohydrate and anoncarbohydrate moiety.

Saccharification enzymes may be obtained commercially. Such enzymesinclude, for example, Spezyme® CP cellulase, Multifect® xylanase,Accelerase® 1500, Accellerase® DUET, and Accellerase® Trio™(Dupont™/Genencor® , Wilmington, Del.), and Novozyme-188 (Novozymes,2880 Bagsvaerd, Denmark). In addition, saccharification enzymes may beunpurified and provided as a cell extract or a whole cell preparation.The enzymes may be produced using recombinant microorganisms that havebeen engineered to express one or more saccharifying enzymes. Forexample, an H3A protein preparation that may be used forsaccharification of pretreated cellulosic biomass is an unpurifiedpreparation of enzymes produced by a genetically engineered strain ofTrichoderma reesei, which includes a combination of cellulases andhemicellulases and is described in WO 2011/038019, which is incorporatedherein by reference.

Chemical saccharification treatments may be used and are known to oneskilled in the art, such as treatment with mineral acids including HCland H₂SO₄ (U.S. Pat. No. 5,580,389; WO2011002660).

Sugars such as glucose, xylose and arabinose are released bysaccharification of lignocellulosic biomass and these monomeric sugarsprovide a carbohydrate source for a biocatalyst used in a fermentationprocess. The sugars are present in a biomass hydrolysate that is used asfermentation medium. The fermentation medium may be composed solely ofhydrolysate, or may include components additional to the hydrolysatesuch as sorbitol or mannitol at a final concentration of about 5 mM asdescribed in U.S. Pat. No. 7,629,156, which is incorporated herein byreference. The biomass hydrolysate typically makes up at least about 50%of the fermentation medium. Typically about 10% of the final volume offermentation broth is seed inoculum containing the biocatalyst. Themedium comprising hydrolysate is fermented in a fermenter, which is anyvessel that holds the hydrolysate fermentation medium and at least onebiocatalyst, and has valves, vents, and/or ports used in managing thefermentation process.

Any biocatalyst that produces a target product utilizing glucose andpreferably also xylose, either naturally or through genetic engineering,may be used for fermentation of the fermentable sugars in the biomasshydrolysate made from lignocellulosic biomass. Target products that maybe produced by fermentation include, for example, acids, alcohols,alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins,peptides, amino acids, vitamins, antibiotics, and pharmaceuticals.

Alcohols include, but are not limited to methanol, ethanol, propanol,isopropanol, butanol, ethylene glycol, propanediol, butanediol,glycerol, erythritol, xylitol, mannitol, and sorbitol. Acids may includeacetic acid, formic acid, lactic acid, propionic acid,3-hydroxypropionic acid, butyric acid, gluconic acid, itaconic acid,citric acid, succinic acid, 3-hydroxyproprionic acid, fumaric acid,maleic acid, and levulinic acid. Amino acids may include glutamic acid,aspartic acid, methionine, lysine, glycine, arginine, threonine,phenylalanine and tyrosine. Additional target products include methane,ethylene, acetone and industrial enzymes.

The fermentation of sugars in biomass hydrolysate to target products maybe carried out by one or more appropriate biocatalysts, that are able togrow in medium containing biomass hydrolysate, in single or multistepfermentations. Biocatalysts may be microorganisms selected frombacteria, filamentous fungi and yeast. Biocatalysts may be wild typemicroorganisms or recombinant microorganisms, and may include, forexample, organisms belonging to the genera of Escherichia, Zymomonas,Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus,and Clostridiuma. Typical examples of biocatalysts include recombinantEscherichia coli, Zymomonas mobilis, Bacillus stearothermophilus,Saccharomyces cerevisiae, Clostridia thermocellum, Thermoanaerobacteriumsaccharolyticum, and Pichia stipitis. To grow well and have high productproduction in a lignocellulosic biomass hydrolysate fermentation broth,a biocatalyst may be selected or engineered to have higher tolerance toinhibitors present in biomass hydrolysate such as acetate. For example,the biocatalyst may produce ethanol as a target product, such asproduction of ethanol by Zymomonas mobilis as described in U.S. Pat. No.8,247,208, which is incorporated herein by reference. Fermentation iscarried out with conditions appropriate for the particular biocatalystused. Adjustments may be made for conditions such as pH, temperature,oxygen content, and mixing. Conditions for fermentation of yeast andbacterial biocatalysts are well known in the art.

In addition, saccharification and fermentation may occur at the sametime in the same vessel, called simultaneous saccharification andfermentation (SSF). In addition, partial saccharification may occurprior to a period of concurrent saccharification and fermentation in aprocess called HSF (hybrid saccharification and fermentation).

For large scale fermentations, typically a smaller culture of thebiocatalyst is first grown, which is called a seed culture. The seedculture is added to the fermentation medium as an inoculum typically inthe range from about 2% to about 20% of the final volume.

Typically fermentation by the biocatalyst produces a fermentation brothcontaining the target product made by the biocatalyst. For example, inan ethanol process the fermentation broth may be a beer containing fromabout 6% to about 10% ethanol. In addition to target product, thefermentation broth contains water, solutes, and solids from thehydrolysate medium and from biocatalyst metabolism of sugars in thehydrolysate medium. Typically the target product is isolated from thefermentation broth producing a depleted broth, which may be called wholestillage. For example, when ethanol is the product, the broth isdistilled, typically using a beer column, to generate an ethanol productstream and a whole stillage. Distillation may be using any conditionsknown to one skilled in the art including at atmospheric or reducedpressure. The distilled ethanol is further passed through arectification column and molecular sieve to recover an ethanol product.The target product may alternatively be removed in a later step such asfrom a solid or liquid fraction after separation of fermentation broth.

The syrup co-product of a lignocellulosic biomass fermentation processis produced from the fermentation broth or depleted fermentation broth.An example of syrup production is disclosed in US20120102823, which isincorporated herein by reference. The broth or depleted broth, such aswhole stillage, is separated into solid and liquid streams, where theliquid stream is called thin stillage. Various filtration devices may beused such as a belt filter, belt press, screw press, drum filter, discfilter, Nutsche filter, filter press or filtering centrifuge. Filtrationmay be aided such as by application of vacuum, pressure, or centrifugalforce. To improve efficiency of filtration, a heat treatment may be usedas disclosed in commonly owned and co-pending US20120178976, which isincorporated herein by reference.

Following liquid/solid separation of a lignocellulosic biomasshydrolysate fermentation broth or depleted broth, the solids fraction,or filter cake (also called wetcake), may be burned to supply energy tothe production process. The filter cake may be dried prior to burning,such as by air drying, to reduce moisture.

A product stream may be removed following liquid/solid filtration of alignocellulosic biomass hydrolysate fermentation broth. For example, theliquid stream may be extracted or distilled to generate a productstream, such as distillation to produce an ethanol product stream and aremaining liquid.

The liquid fraction is further purified by evaporation producing waterthat may be recycled and a syrup. Prior to evaporation, a portion of theliquid fraction may be recycled for use as back set, which may be addedat any point in the process where water is needed, such as inpretreatment, saccharification, or biocatalyst seed production.Evaporation may be in any evaporation system, such as falling film,rising film, forced circulation, plate or mechanical and thermal vaporrecompression systems. Evaporation may be continuous or batch and mayuse a multi-effect evaporator. The evaporated water may be recycled inthe overall lignocellulosic biomass hydrolysate fermentation process.The remaining material after evaporation is a syrup which is the presentlignocellulosic syrup. In one embodiment the lignocellulosic syrupcomposition contains from about 40% to about 60% solids or from about40% to about 70% solids (may have about 40%, 45%, 50%, 55%, 60%, 65%, or70% solids), from about 10 g/l to 30 g/l of acetamide, at least about 40g/l of sugars, a density of about 1 to about 2 g/cm³, and viscosity lessthan 500 SSU at 100° F. (38° C.). “SSU” is Saybolt Universal Viscosityin Seconds(Burger V L., Encycl. Ind. Chem. Anal. (1966), Volume 3,768-74). The extent of evaporation may be modulated to achieve thedesired solids content. When the pretreatment process used to preparethe biomass for saccharification is a process that uses ammonia, thelignocellulosic syrup contains at least about 5 g/l of ammonia.

Soil Conditioning Material

A soil conditioning material is combined with the presentlignocellulosic syrup to produce the present agricultural composition.

A soil conditioning material is a substance that when applied to soil,improves the properties of the soil such that at least one of plantgrowth and yield are increased. Soil properties that may be improvedinclude, but are not limited to, pH, drainage, providing plantnutrients, soil structure, cation exchange capacity, and waterretention. Any soil conditioning material that is mixable may be used inthe present invention. Typically the soil conditioning material used isa material that is particulate, and is powdery, dusty, or granular. Inone embodiment the soil conditioning material is in the form of apowder.

In various embodiments the additional fuel component is lime or gypsum.Lime that is used for soil application is typically crushed or groundlimestone which is made of calcium carbonate. Chalk is a form oflimestone that is a soft, white, porous sedimentary rock. Dolomitic limeis a type of lime that contains calcium carbonate and magnesiumcarbonate. Lime is alkaline and is used to neutralize acidity of soiland add calcium, and may add magnesium (if using dolomitic lime). Gypsumis hydrated calcium sulfate. Gypsum removes sodium from soil andreplaces it with calcium thereby reducing salinity of soli, andchemically loosens clay soil by competing with salt in the clay therebyimproving drainage and plant root growth.

Agricultural Composition

The present agricultural composition contains the lignocellulosic syrupdescribed herein and a soil conditioning material, as described above.The lignocellulosic syrup and soil conditioning material are combined inamounts wherein the syrup acts as a binder of the soil conditioningmaterial, which is typically crushed or powdered. In various embodimentsthe syrup is between about 2% and about 20% by weight of the finalcombination of syrup and soil conditioning material. The syrup may beabout 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, or 20% of the total combined weight. In variousembodiments the syrup is between about 2% and 20%, or between about 2%and 10%, of the final composition of syrup and soil conditioningmaterial, and the soil conditioning material is lime, gypsum, or acombination thereof.

In the present process for producing an agricultural composition, thelignocellulosic syrup and soil conditioning material are combined in aratio that is between about 1:50 and about 1:5. The ratio of thelignocellulosic syrup and soil conditioning material may be betweenabout 1:50 and 1:6.7, or between about 1:50 and 1:10. The combination oflignocellulosic syrup and soil conditioning material forms into aconveniently handled solid material. Various shapes may be formed suchas pellets, granules, irregular shapes, and the like. In one embodimentthe syrup is sprayed over the soil conditioning material in a rotatingdrum as it rotates. In one embodiment the lignocellulosic syrup and soilconditioning material composition is dried. Alternative processing mayinclude treatments such as heating, compressing, extruding, pelleting,molding, and/or drying.

Use Of Agricultural Composition

The present agricultural composition is applied to soil by any methodknown to one skilled in the art. For example, the agriculturalcomposition may be applied by spreading using a conventional fertilizerspreader of any size, such as for lawn care or for agricultural fields.The agricultural composition may be tilled into the soil prior toplanting, applied after planting, and/or applied periodically during thegrowing season.

Typically soil testing is performed prior to application to determinethe specific type and amount of agricultural composition to be appliedto soil.

EXAMPLES

The present invention is further defined in the following Examples.

It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations used is as follows: “s” is second, “min”means minute(s), “h” of “hr” means hour(s), “μL” or “μl” meansmicroliter(s), “mL” or “ml” means milliliter(s), “L” or “l” meansliter(s), “m” is meter, “nm” means nanometer(s), “mm” meansmillimeter(s), “cm” means centimeter(s), “μm” means micrometer(s), “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “μmole”means micromole(s), “g” means gram(s), “μg” means microgram(s), “mg”means milligram(s), “kg” is kilogram, “rpm” means revolutions perminute, “C” is Centigrade, “ppm” means parts per million, “cP” iscentipoise, “g/l” means grams per liter, “SSU” is Saybolt UniversalViscosity in Seconds, “wt %” is weight %.

General Methods: Saccharification Enzymes

Accellerase® 1500 (A1500) and Multifect® Xylanase are obtained fromDanisco U.S. Inc., Genencor, International (Rochester, N.Y.).

Cellulase and Hemicellulase Production Strain

Strain 229: A Trichoderma reesei strain, derived from RL-P37(Sheir-Neiss and Montenecourt, 1984, Appl. Microbiol. Biotechnol.20:46-53) through mutagenesis and selection for high cellulaseproduction, was co-transformed with the β-glucosidase expressioncassette (cbh1 promoter, T. reesei β-glucosidase) gene, cbh1 terminator,and amdS marker), and the endoxylanase expression cassette (cbh1promoter, T. reesei xyn3, and cbh1 terminator) using PEG mediatedtransformation (Penttila et al., 1987, Gene 61(2):155-64). Numeroustransformants were isolated and examined for R-glucosidase andendoxylanase production. One transformant, referred to as T. reeseistrain #229, was used in certain studies described herein.

Strain H3A: T. reesei strain #229 was co-transformed with theβ-xylosidase Fv3A expression cassette (cbh1 promoter, Fv3A gene, cbh1terminator, and alsR marker), the β-xylosidase Fv43D expression cassette(egl1 promoter, Fv43D gene, native Fv43D terminator), and the Fv51Aα-arabinofuranosidase expression cassette (egl1 promoter, Fv51A gene,Fv51A native terminator) using electroporation. Transformants wereselected on Vogels agar plates containing chlorimuron ethyl. Numeroustransformants were isolated and examined for β-xylosidase andL-α-arabinofuranosidase production. T. reesei integrated expressionstrain H3A, which recombinantly expresses T. reesei β-glucosidase 1, T.reesei xyn3, Fv3A, Fv51A, and Fv43D was isolated.”

Extra cellular protein produced during fermentation of strain H3A wasseparated from the cell mass by centrifugation, concentrated bymembrane-ultrafiltration through a Millipore 10 kD molecular cut offweight membrane and pH adjusted to 4.8. Total protein was determinedusing a modified Biuret method as modified by Weichselbaum and Gornallusing Bovine Serum Albumin as a calibrator (Weichselbaum, 1960, Amer. J.Clin. Path. 16:40; Gornall et al., 1949 J. Biol. Chem 177:752). This H3Aextracellular protein preparation, called herein H3A protein, was usedas a combination cellulase and hemicellulase preparation effectingcomplex carbohydrate hydrolysis during SSF.

Biocatalyst and Inoculum Preparation

Origin of the Zymomonas mobilis Strains for Fermentation

A lignocellulosic biomass hydrolysate fermentation broth may be madeusing alternative biocatalysts. Exemplary strains are are describedbelow. As an alternative, strain ZW658, deposited as ATCC #PTA-7858, maybe used to produce a lignocellulosic biomass hydrolysate fermentationbroth for processing.

Zymomonas mobilis strain ZW705 was produced from strain ZW801-4 by themethods detailed in U.S. Pat. No. 8 247 208, which is hereinincorporated by reference, as briefly restated here. Cultures of Z.mobilis strain ZW801-4 were grown under conditions of stress as follows.ZW801-4 is a recombinant xylose-utilizing strain of Z. mobilis that wasdescribed in U.S. Pat. No. 7,741,119, which is herein incorporated byreference. Strain ZW801-4 was derived from strain ZW800, which wasderived from strain ZW658, all as described in U.S. Pat. No. 7,741,119.ZW658 was constructed by integrating two operons, PgapxylAB andPgaptaltkt, containing four xylose-utilizing genes encoding xyloseisomerase, xylulokinase, transaldolase and transketolase, into thegenome of ZW1 (ATCC #31821) via sequential transposition events, andfollowed by adaptation on selective media containing xylose. ZW658 wasdeposited as ATCC #PTA-7858. In ZW658, the gene encodingglucose-fructose oxidoreductase was insertionally-inactivated usinghost-mediated, double-crossover, homologous recombination andspectinomycin resistance as a selectable marker to create ZW800. Thespectinomycin resistance marker, which was bounded by loxP sites, wasremoved by site specific recombination using Cre recombinase to createZW801-4.

A continuous culture of ZW801-4 was run in 250 ml stirred, pH andtemperature controlled fermentors (Sixfors; Bottmingen, Switzerland).The basal medium for fermentation was 5 g/L yeast extract, 15 mMammonium phosphate, 1 g/L magnesium sulfate, 10 mM sorbitol, 50 g/Lxylose and 50 g/L glucose. Adaptation to growth in the presence of highconcentrations of acetate and ammonia was effected by graduallyincreasing the concentration of ammonium acetate added to the abovecontinuous culture media while maintaining an established growth rate asmeasured by the specific dilution rate over a period of 97 days.Ammonium acetate was increased to a concentration of 160 mM. Furtherincreases in ammonium ion concentration were achieved by addition ofammonium phosphate to a final total ammonium ion concentration of 210 mMby the end of 139 days of continuous culture. Strain ZW705 was isolatedfrom the adapted population by plating to single colonies andamplification of one chosen colony.

Strain AR3 7-31 was produced from strain ZW705 by further adaptation forgrowth in corn cob hydrolysate medium as disclosed in U.S. Pat. No.8,476,048, which is incorporated herein by reference. ZW705 was grown ina turbidostat (U.S. Pat. No. 6,686,194; Heurisko USA, Inc. Newark,Del.), which is a continuous flow culture device where the concentrationof cells in the culture was kept constant by controlling the flow ofmedium into the culture, such that the turbidity of the culture was keptwithin specified narrow limits. Two media were available to the growingculture in the continuous culture device, a resting medium (Medium A)and a challenge medium (Medium B). A culture was grown on resting mediumin a growth chamber to a turbidity set point and then was diluted at adilution rate set to maintain that cell density. Dilution was performedby adding media at a defined volume once every 10 minutes. When theturbidostat entered a media challenge mode, the choice of addingchallenge medium or resting medium was made based on the rate of returnto the set point after the previous media addition. The steady stateconcentration of medium in the growth chamber was a mix of Medium A andMedium B, with the proportions of the two media dependent upon the rateof draw from each medium that allowed maintenance of the set celldensity at the set dilution rate. A sample of cells representative ofthe population in the growth chamber was recovered from the outflow ofthe turbidostat (in a trap chamber) at weekly intervals. The cell samplewas grown once in MRM3G6 medium and saved as a glycerol stock at −80° C.

ZW705 was grown to an arbitrary turbidity set point that dictated thatthe culture use all of the glucose and approximately half of the xylosepresent in the incoming media to meet the set point cell density at theset dilution rate. Using resting medium that was 50% HYAc/YE and 50%MRM3G6.5X4.5NH₄Ac12.3 and challenge medium that was HYAc/YE. A strainisolated after 3 weeks was used in another round of turbidostatadaptation using HYAc/YE as the resting medium and HYAc/YE+9 weight %ethanol as the challenge medium. Strain AR3 7-31 was isolated after 2weeks and was characterized as a strain with improved xylose and glucoseutilization, as well as improved ethanol production, in hydrolysatemedium. By sequence analysis, AR3 7-31 was found to have a mutation inthe Zymomonas mobilis genome ORF encoding a protein havingcharacteristics of a membrane transport protein, and annotated asencoding a fusaric acid resistance protein.

Media

-   -   MRM3 contains per liter: yeast extract (10 g), KH₂PO₄ (2 g) and        MgSO₄.7H₂O (1 g)    -   MRM3G6 contains is MRM3 containing 60 g/L glucose    -   MRM3G6.5X4.5NH₄Ac12.3 is MRM3 containing 65 g/L glucose, 45 g/L        xylose, 12.3 g/L ammonium acetate    -   HYAc/YE contains cob hydrolysate from which solids were removed        by centrifugation and that was filter sterilized containing 68        g/L glucose, 46 g/L xylose and 5 g/L acetate, supplemented with        6.2 g/L ammonium acetate and 0.5% yeast extract, adjusted to        pH5.8.

Lignocellulosic Biomass Processing and Fermentation

Corn stover is milled to 3/8″ (0.95 cm). Pretreatment is at 140° C. with14% NH3 and 65% solids for 60 min. Saccharification is at 47° C., pH5.3, with 7.8 mg/g glucan+xylan of an enzyme consortium, for 96 hr.Saccharification enzymes are a mix of cellulases and hemicellulasesexpressed in a Trichoderma reesei strain H3A as described above. Theresulting hydrolysate is used in fermentation. 10 mM sorbitol is addedto the hydrolysate making the fermentation medium, and the pH isadjusted to 5.8.

For the seed, first frozen strain Zymomonas mobilis AR3 7-31 stock isgrown in MRM3G6 (10 g/L BBL yeast extract, 2 g/L KH₂PO₄, 1 g/LMgSO₄*7H₂O, 60 g/L glucose) at 33° C., without shaking for 8 hr as arevival culture. MRM3G10 medium (same as MRM3G6 but with 100 g/Lglucose) is inoculated with revival culture, and incubated at 33° C.with shaking for 14-16 hr. Growth is to an OD₆₀₀ between 1.5 and 3.1.The entire culture is used to inoculate a seed fermenter to an initialOD₆₀₀ of approximately 0.05.

The seed fermentation is carried out in 10 g/L yeast extract, 2 g/LKH₂PO₄, 5 g/L MgSO₄*7H₂O, 10 mM sorbitol, and 150 g/L glucose. Seedfermentation is performed at 33° C. and pH 5.5. Seed is harvested afterfirst observation of glucose reduction to less than 50 g/L, with glucosemeasured by using a YSI 2700 SELECT™ Biochemistry Analyzer (YSI LifeSciences; Yellow Springs, Ohio).

The seed is added to the hydrolysate medium in the fermenter.Fermentations are carried out at 30° C.-33° C. for 48-72 hr.

Lignocellulosic Syrup

The fermentation broth is distilled to recover ethanol and the remainingwhole stillage is filtered. The liquid fraction is passed throughevaporators removing overhead water, and producing a syrup.

Example 1 Production of Limestone Pellets from Ground Limestone andLignocellulosic Syrup

Raw limestone mined from a quarry in Weeping Water, Nebraska is groundinto a fine powder and pneumatically fed into a rotary drum. Syrupproduced from a lignocellulosic biomass ethanol production process (seeGeneral Methods) is sprayed into the drum filled with the groundlimestone as it is rotated. Batches are prepared using syrup as 2 to 10wt % of the total weight of ground limestone and syrup. The resultingmixture is fed to a dryer and the resulting pellets are allowed to cooland harden. The limestone powder is held together in the resulting solidpellets, with little evidence of powdery material.

Example 2 Production of Gypsum Pellets from Ground Gypsum andLiqnocellulosic Syrup

Raw gypsum mined from a quarry in Fort Dodge, Iowa is ground into a finepowder and pneumatically fed into a rotary drum. Syrup produced from alignocellulosic biomass ethanol production process (see General Methods)is sprayed into the drum filled with ground gypsum as it is rotated.Batches are prepared using syrup as 2 to 10 wt % of the total weight ofground gypsum and syrup. The resulting mixture is fed to a dryer and theresulting pellets are allowed to cool and harden. The gypsum powder isheld together in the resulting solid pellets, with little evidence ofpowdery material.

What is claimed is:
 1. An agricultural composition comprising: a) alignocellulosic syrup; and b) at least one soil conditioning material.2. The agricultural composition of claim 1 wherein the syrup is aco-product of a process for the production of alcohol from alignocellulosic biomass.
 3. The agricultural composition of claim 1wherein the syrup comprises: a) from about 40% to about 70% solids; b)from about 10 g/l to about 30 g/l of acetamide; and c) at least about 40g/l of sugars; wherein the cellulosic syrup has a density of about 1 toabout 2 g/cm3 and a viscosity of less than 500 SSU at 100° F. (38° C.).4. The agricultural composition of claim 2 wherein the process for theproduction of alcohol from a cellulosic biomass uses ammonia for thepretreatment of the lignocellulosic biomass, and the syrup contains atleast about 5 g/l of ammonia.
 5. The agricultural composition of claim 2wherein the lignocellulosic biomass is selected from the groupconsisting of corn cobs, corn stover, grasses, wheat straw, barleystraw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse,sorghum plant material, soybean plant material, woody plants,vegetables, fruits, flowers, empty palm fruit bunch, and energy cane. 6.The agricultural composition of claim 1 wherein the at least one soilconditioning material is selected from the group consisting of lime,gypsum, and combinations thereof.
 7. The agricultural composition ofclaim 1 wherein the at least one soil conditioning material is in theform of a powder.
 8. The agricultural composition of claim 1 wherein thesyrup is between about 2% and about 20% of the composition, by weight.9. The agricultural composition of claim 1 wherein the composition is inthe form of pellets or granules.
 10. A process for the production of anagricultural composition comprising combining the lignocellulosic syrupof claim 1 with at least one soil conditioning material, wherein pelletsor granules comprising the syrup and the conditioning material areproduced.
 11. The process of claim 10 wherein the syrup is a co-productof a process for the production of alcohol from a lignocellulosicbiomass.
 12. The process of claim 10 wherein the syrup comprises: a)from about 40% to about 70% solids; b) from about 10 g/l to about 30 g/lof acetamide; and c) at least about 40 g/l of sugars; wherein thecellulosic syrup has a density of about 1 to about 2 g/cm3 and aviscosity of less than 500 SSU at 100° F. (38° C.).
 13. The process ofclaim 10 wherein the soil conditioning material is selected from lime,gypsum, or a combination thereof.
 14. The process of claim 10 whereinthe syrup is sprayed over the soil conditioning material in a rotatingdrum.
 15. The process of claim 10 wherein the pellets or granules aredried.
 16. The process of claim 10 wherein the lignocellulosic syrup andthe soil conditioning material are combined in a ratio that is betweenabout 1:50 and about 1:5.
 17. A method for conditioning soil comprisingapplying the agricultural composition of claim 1 to the soil.